Meaningful molecular analysis of single mammalian cells by submicron focused laser desorption mass spectrometry has been an elusive goal. However, such microanalysis has been achieved with our unique MALDI-Ion Mobility-orthogonal Time of Flight Mass Spectrometry (MALDI-IM-oTOFMS). Applying submonolayers of special nanoparticulate matrices (NP) onto sub cellular tissue volumes enables a micro focused laser to desorb neutral analyte molecules (which are then post-ionized with a second laser and mass analyzed). Thus, the applicants'main goal now is the creation and optimization of new nanoparticulate matrices and techniques for rapid dissemination of NPs into sub cellular volumes. We will (1) demonstrate sub cellular molecular profiling by NP implantation and compare against matrices used in MALDI tissue imaging (2) design nanoparticulates which promote the non-destructive laser desorption of neutral analytes which can then be post-ionized with a second laser, and (3) demonstrate molecular depth profiling of cells through repeated cycles of matrix implantation followed by molecular analysis by MALDI-IM-oTOFMS (with post- ionization). This approach will be compared with several standard histological stains for DNA, RNA and f-actin (among others). We consider these developments to be critical bridging technologies for transforming quantitative micro-analytical methods into biochemical imaging of sub cellular molecular structure useful for basic research and pathological analysis. PUBLIC HEALTH RELEVANCE: The Ionwerks nanoparticulate implanter is now offered for sale, and the innovations described by the applicants can be rapidly tested and integrated to augment the power of an already useful instrument and technique which is in its commercial infancy. The rapid implantation of nanoparticulate matrices is essential technology for intracellular analysis and intracellular analysis is the basis for molecular phenotyping. Thus achieving cellular level mass spectrometry-based molecular phenotyping seems feasible and would be an important development in biomedical sciences and clinical pathology. Simple approaches, such as overlays of the mass spec image and light (or confocal) microscopic images should provide fast, streamlined, broad molecular phenotyping of even small or limited biopsy samples.